Advance Journal of Food Science and Technology 2(5): 298-302, 2010
ISSN: 2042-4876
© M axwell Scientific Organization, 2010
Submitted Date: August 12, 2010
Accepted Date: September 13, 2010
Published Date: September 25, 2010
Thin Layer Chromatography Methods for Rapid Identity Testing
of Morinda citrifolia L. (Noni) Fruit and Leaf
Brett J. West and Shixin Deng
Research and Development Department, Tahitian Noni International, 737 East, 1180 South,
American Fork, Utah, USA, 84003
Abstract: Mo rinda citrifolia L., com mon ly known as no ni, is a grow ing glo bal comm odity. A s such , there is
a need for rapid and inexpensive identity tests of n oni fruit and leaf products. Thin Layer Chromatography
(TLC) methods w ere develop ed for the identification of deacetylasp erulosidic acid in noni fruit and leaf
products. TLC methods were also developed for the identification of scopoletin in noni fruit products and rutin
in noni leaf produ cts. TLC results were su pported by High P erformanc e Liquid C hromatography (HPLC)
analyses. The concentrations of marker compounds detected by HPLC indicate that these TLC methods have
good sensitivity and utility in the identification of noni fruit and leaf ingredients from a range of global sources,
as well as noni based comme rcial products. These methods do no t require expensive instrumentation or
specialized laboratories, and are readily transferable to laboratories op erating under a variety of circumstances.
Key w ords: Identity testing, Mo rinda citrifolia, thin layer chromatography
INTRODUCTION
Morinda citrifolia Linnaeus, noni, fruit juice and
powder are growing global commod ities. In recent years,
noni fruit puree and juice comprised 22 to 46% of total
agricultural exports from French Polyne sia to the United
States (Service du Comm erce E xterieur de Po lynesie
Frances, 2008a). In 2005 alone, more than 6,000 metric
tons of noni fruit puree was produced (Service du
Commerce Exterieur de Polynesie Frances, 2008b). Since
2004, noni has been Samoa’s major agricultural ex port,
with a peak annual export of approximately 1.5 million
liters of juice and 167 metric tons of dried fruit
(Roge rs et al., 2010). In 2003, Noni fruit juice was
approved by the European Union as a novel food
ingredient to be used in pasteurized fruit beverages
(European Comm ission, 2003). That approval was based
on the assessm ent of noni fruit juice from French
Polynesia (Scientific Committee on Food, 2002). Since
that approval, additional sources of noni fruit juice have
been approved for sale within the EU under the
substantial equivalence application procedure. More than
40 substantial equiv alence app rovals within the EU have
been granted. These sources are from a variety of nations
which include French Polyensia, Fiji, Dominican
Republic, Panam a, Costa R ica, Samo a, U.S.A . (Haw aii),
Tonga, Vanuatu, Co ok Islands, Palau, Solomon Islands,
and Nauru (European Commission, 2010a). Additionally,
noni leaves were approved in 2008 as a novel food within
the EU, for the purpose of making infusions (European
Commission, 2008). Very recently, noni fruit puree and
noni fruit juice concentrate received approval as novel
food ingredients to be used in a variety of food product
categories (E uropean C omm ission, 2010b).
The increasing production and so urces of non i fruit
and leaves necessitates stand ardization regarding identity
testing. Analyses of commercial noni products have
indicated some quality differences, as well as po ssible
misidentification of
noni
fruit
raw
materials
(West et al., 2006; European Food Safety Authority,
2008; Deng et al., 2010a). Methods have been published
that might be useful for identity testing, but these require
expensive instrumentation and are costly to perform.
There is a need for simple, acurate and cost effective
methods for authenticating noni fruit and leaf materials.
This is especially true when considering the sources of
noni, which are often developing countries. Many
producers do not have the means to conduct testing which
requires investment in costly equipment. Additionally,
rapid testing needs to be available to governmental
inspectors around the world to confirm the identity of
noni fruit and leaves that are imported into their
respective nations. To address the need for low cost, yet
acurate, analytical methods for identifying authentic noni
fruit and leaf raw material and commercial products, Thin
Layer Chromatography (TLC) methods were developed.
TLC is a widely used analytical method for qua lity
control in food production and agriculture. It is used to
detect a wide range of plant constituents (Sherma , 2000).
The accuracies of the current TLC method s were
confirmed by High Performance Liquid Chromatography
(HPL C). The newly developed TLC methods can be
Corresponding Author: Brett J. West, Research and Development Department, Tahitian Noni International, 737 East, 1180 South,
American Fork, Utah, USA, 84003. Tel: +1 801 234 3621; Fax: +1 801 234 1030
298
Adv. J. Food Sci. Technol., 2(5): 298-302, 2010
performed in unspecialized laboratories and do not require
any expensive analytical instrumentation. Chemical
reference standards may be employed in the assay, or
certified reference plant materials may be used. The use
of certified n oni reference material makes this method
readily accessible to most quality control departments and
governm ental food inspection age ncies.
extracts were redissolved with 5 mL of MeO H. Voucher
specimens of all samples are deposited in our laboratory.
Analysis of the deacetylasperulosidic acid content of
the samples was performed by high perform ance liquid
chromatography (HPLC ), according to a previously
reported method (Deng, 2010). Extracted solid samples
and juices were dissolved in solvents, mixed thoroughly
and then filtered through a 0.2 :m PTFE filter for HPLC
analysis. Deacety lasperulosidic acid (DAA) was
accu rately weighed and then dissolved in an appro priate
volume of MeO H to produce corresponding standard
solutions rangin g from 0.00174 to 1.74 mg/mL.
Calibration curves of the standards were plotted after
linear regression of the peak areas versus concentrations.
Chromatogra phic separation was performed on a W aters
2690 separations module coupled with 996 PDA
detectors, equipped with a C18 column. Elution was
accomplished with tw o mo bile phases, MeCN, and 0.1%
formic acid in H 2 O (v/v), with a flow rate of 0.8 mL/min.
A linear gradien t of 100 % aqueous fo rmic acid (0.1%) for
0-5 min, followed by 70 % aqueous fo rmic acid and 30%
MeCN for 40 m in, wa s used . The PDA detector was
monitored in the range of 210-400 nm. The injection
volume was 10 :L for each of the sample solutions. The
colum n temperatu re was maintained at 25ºC.
Analysis of scopoletin and rutin content of the
samples was also performed by HPLC, according to a
previously reported method (Deng et al., 2010a).
Chemical standards of scopoletin and rutin were
accu rately weighed and then dissolved in an appro priate
volume of MeOH/MeCN to produce corresponding stock
standard solutions. Working standard solutions for
calibration curves were prepared by diluting the stock
solutions with MeOH at different concentrations. A ll
stock and working solutions were maintained at 0ºC in a
refrigerator. Samples were extracted and dissolv ed in
MeOH. Chromatographic separation was performed on a
W aters 2690 separations module coupled with 996 a
photodiode array (PDA ) detector, and equipped with a
C18 column. The mob ile phase system was composed of
three solvents: A; MeCN, B ; MeOH and C ; 0.1 % TFA in
H 2 O (v/v). The mobile phase was programmed
consecutively in linear gradients as follows: 0 min, 10%
A, 10% B, and 80% C; 15 min, 20% A, 20% B, and 60%
C; 26 min, 40% A, 40% B, and 20% C; 28–39 min, 50%
A, 50% B, and 0% C; and 40-45 min, 10% A, 10% B, and
80% C. The elution was run at a flow rate of 1.0 mL/min.
The UV spectra we re qua ntified at 3 65 nm.
Table 1 summarizes the mobile phases and
visualization methods used in the identification of each
chemical marker. Sample solutions (5 :L each ) were
spotted onto silica gel 60 F254 TLC plates (Merck,
Darmstadt, Germany), and developed with the three
mob ile phases. Visualization of the compounds was
performed with either UV lamps or coloring spray
reage nt.
MATERIALS AND METHODS
Chemical marker standards, scopoletin, rutin, and
deac etylasp erulosidic acid (DA A), were isolated directly
from the freeze-dried noni fruit and leaf pow der. Identity
and purity (>98%) were confirmed by NM R, MS, and
HPLC (Deng et al., 2007, 2010a, b). The marker
compounds were dissolved in methanol (MeOH ) to a
concentration of 1 mg/mL.
Eight raw noni fruit samples (F1-F8) were collected
from different location s includ ing French Polynesia
(Tahiti, Moorea, and Motu Fareone), Tonga, Dominican
Republic, Okinawa, Thailand, and H awaii. The fruit
samples were stored below 0ºC before use. Fruits w ere
thawed, mashe d and ex tracted (2 g) twice with 125 mL
methanol (MeOH), aided by sonication for 30 min. The
solvent was rem oved under vacuum in a rotary
evaporator. The MeOH extracts w ere then redissolved in
10 m L of M eOH.
Commercial noni fruit juice products (J1-J4),
originating from Tahiti, Dominican Republic, Hawaii, and
Costa Rica, produced by different m anufacturers, w ere
purchased at local m arkets or via the internet. Prior to
analysis, all sample s were filtered through a 0.45 :m
nylon membrane filter and then purified by Solid-Phase
Extraction (SPE) with Waters OASISS® extraction
cartridges. SPE cartridges w ere first equilibrated w ith
water, followed by methanol. The samples were then
loaded onto the cartridge and washed with 5% MeOH,
followed by 100% MeOH. The MeO H eluate was retained
for TLC analysis.
Four different no ni fruit powder capsule products
were also purchase. The pro ducers of the capsules are
located in French Polynesia (C1), Hainan, South China
Sea (C2), Haw aii (C3), and Indonesia (C4). One gram of
the capsule contents was extracted with 5 mL MeOH,
aided by sonication for 10 min. T he M eOH extracts were
filtered, and the solvent removed by evaporation under
vacuum at 50ºC. The extract was then redissolved in 1 mL
of M eOH.
Noni leaves were collected from French Polynesia
(L1), Tonga (L2), Panama (L3), and Saipan, Northern
Mariana Islands (L4). These samples were air-dried. Two
grams of each sample were extracted with 100 mL of
ethanol (EtOH) in a sonicator for 30 min. The extractants
were filtered and the solvent removed by evap oration in
a rotary evaporator under vacuum at 45ºC. The EtOH
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Adv. J. Food Sci. Technol., 2(5): 298-302, 2010
Table 1: TLC method parameters for chemical markers of noni fruit and leaves
Chemical marker
Mo bile phase
Visualization method
De acety laspe rulos idic a cid
dichloromethane: methanol: water
spray reagent: 2% anisaldehyde, 10%
(13: 6: 1, v:v:v)
s ul fu ri c a ci d i n E tO H
Sco pole tin
dichloromethane: methanol
UV lamp, 365 nm
(19: 1, v:v)
Ru tin
ethylacetate: formic acid: water
UV lamp, 254 nm
(7: 1.5: 1.7, v:v:v)
Sp ot ch aracte ristic
blue colored spot
fluorescent light blue spot
dark spo t
This study was conducted during 2010 at the
laboratories of Tahitian Noni International, American
Fork, Utah , and U .S.A.
RESULTS AND DISCUSSION
Results of the HPLC analyses indicate a wide range
in the concentrations of marker compounds among the
various sources of noni fruit and leaves, and resulting
commercial products. The analyses confirmed the
presence of deacetylasperulosidic acid in all samples, as
well as scopoletin in all noni fruit based samples. Rutin
was detected in all leaf samples. The DAA content of the
MeOH extract of raw noni fruit sa mple s wa s 13.8-42.9
mg/g, while scop oletin was prese nt in the ra nge of 0.7-6 .9
mg/g. Noni fruit powder capsules contained 7.2-11.8 mg
DA A/g and 0.1-0.4 mg scopoletin/g. DAA and scopoletin
occurred in commercial noni juice products at 0.2-1.7
mg/mL and 3.7-21.2 :g/mL, respectively. Dried noni leaf
samples contained DA A and rutin in the range of 0 .9-3.1
mg/g and 0.4-3.6 mg/g, respectively.
In the scopoletin analyses, fluo rescent light blu e spots
were visible to the unaided eye on the developed TLC
plates, when viewed under long wave UV light, 365 nm.
The scopoletin standard produced the most intensely
fluorescent light blue spot. Every noni fruit, juice, and
capsule also produced the sam e spot with a retention
factor (Rf) of 0.5, but with differing intensities (Fig. 1
and 2). Scopoletin was also detected by this TLC method
at the lowest concentration in any sample, w hich w as 3.7
:g/mL found in sample J3 (Fig. 1). At this low
concentration, a faintly fluorescent light blue spot was
still visible.
Rutin was visible on the developed TLC plate under
short wave UV light, 254 nm, w ith an Rf of 0.5. At this
wavelength, the silica g el will fluoresce green, whereas
the rutin absorbs the light and appears as a dark spot.
Under the conditions of this ana lysis, rutin was visible in
all noni leaf samples (Fig. 3). The lowest concentration
visualized in these analyses, as confirmed by the HPLC
analy ses, w as 0.4 mg/g .
Upon heating, visualization of the DAA standard was
accomplished with the 2% anisald ehyde spray reagent. A
blue color developed, according to results with similar
iridoid glycosides (Harborne, 1998). Blue color
developm ent, upon treatm ent w ith the an isaldehyde
reagent and heating, also occurred with all samples,
regardless of origin or form, with an Rf of 0.3
Fig. 1: Scopoletin analysis of commercial noni juice and noni
capsules by TLC. C1-C4: commercial noni capsules
from different manufacturers; J1-4: commercial noni
fruit juices from different manufacturers. S: scopoletin
Fig. 2: Scopoletin analysis of noni fruit by TLC. F1-F8: noni
fruit samples from different regions. S: scopoletin
Fig. 3: Rutin analysis of noni leaf by TLC. L1-L4: noni leaf
samples from different regions. R: rutin
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Adv. J. Food Sci. Technol., 2(5): 298-302, 2010
Fig. 4: Deacetylasperulosidic acid analysis of noni fruit and leaves by TLC. F1-F8: noni fruit samples from different regions. L1-L4:
noni leaf samples from different regions. DAA: deacetylasperulosidic acid
Fig. 5: Deacetylasperulosidic acid analysis of commercial noni juice and noni capsules by TLC. C1-C4: commercial noni capsules
from different manufacturers; J1-4: commercial noni fruit juices from different manufacturers. DAA: deacetylasperulosidic
acid
(Fig. 4 and 5). The com merc ial noni juice samples
represent the lowest concentration ranges for DAA . Blue
color deve lopm ent w as distinctive also at the lowest
concentration, 0.2 mg/mL.
Two com pounds in noni fruit and two in noni leaves
are useful chemical markers for identification purposes.
For noni fruit, scopoletin and deac etylasperuloside were
chosen as markers. Scopoletin has been found in
authe ntic noni fruit samples from all tropical regions
around the globe and has been identified in many
commercial
noni
juice
products,
as
well
(Deng et al., 2010a). Iridoid glycosides are
chem otaxo nom ic markers for plants in the Rubiaceae
family (Inouye, 19 88). D eacetylasperulosidic acid has
been found to be the major iridoid glycoside, as well as
the major phytochemical constituent, of noni fruit
(Deng, 2010; Potterat et al., 2007; Kamiya et al., 2005).
Deacetylasperulosidic acid is also a useful chemical
marker for noni leaves (Sang et al., 2001). Rutin has been
isolated and identified as a major flavonoid in the leaves
(Deng et al., 2007 ), and, therefore , rutin was chosen as the
second chemical marker for noni leaf products.
All TLC analysis results w ere confirmed by the resu lt
of the HPLC analyses. The sensitivity of the scopoletin
TLC method is appa rently very go od, since as little as 3.7
:g/mL produced a fluorescent blue spot that was
discernable to the un aided eye. Rutin and DAA content
ranges were mu ch hig her than scopoletin, but still
demonstrated good sensitivities at 0.4 mg /g and 0.2
mg/mL, respectively. Q uantities lower than these are
possible in some commercial products where noni
ingred ients are blended with others. However, detection
in these products can be enhanced by concentration of the
extracts produced during sample preparation.
The utility of the TLC methods for the identification
of noni fruit and leaf ingredients, as well as noni based
commercial products, has been demonstrated. These
methods do not require expensive instrumentation or
specialized laboratories. Therefore, the methods are
readily transferable to analysts working under a variety of
circumstances. The marker compounds utilized in these
methods are characteristic of noni fruit and leaves from all
regions where this plant is cultivated. As DAA is the
major phytoche mical in noni fruit, it is an especially
useful marker for identity testing of the growing number
of noni juice products being sold world-wide.
CONCLUSION
The two iridoids, deacetylasperulosidic acid and
asperulosidic acid, have b een identified in noni fruit and
leaf samples from different geo graphical regions. As such,
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Adv. J. Food Sci. Technol., 2(5): 298-302, 2010
these two chemicals serve as useful identity markers for
authen tic noni fruit and leaf products. HPLC analysis
confirms that the thin layer chromatography method
described herein is a simple, accurate, and economic tool
for performing identity testing of noni fruit and leaf
products. This method can also be readily transferred to
unsp ecialized labo ratories in deve loping nations.
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ACKNOWLEDGMENT
This research was supported by M orinda Holdings,
Inc., C. Jarakae Jensen and ‘Afa K. Palu assisted in
sample collection and plant identification.
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